Serena Williams withdrew from the Italian Open on Tuesday with a recurring left knee injury handing sister Venus a walkover into the third round.
In Tuesday’s statement, the 23-time Grand Slam winner insisted she would still be competing in the French Open later this month.
“I must withdraw from the Italian Open due to pain in my left knee,” Serena said in a statement.
“I will miss the fans and competition at one of my favourite tournaments.
“I’ll be concentrating on rehab and look forward to seeing you all at the French Open and next year in Rome.”
The French Open starts on May 26.
Serena’s withdrawal comes a day after she beat Sweden’s Rebecca Peterson in straight sets to set up a second-round meeting with her older sister Venus.
She had been returning after more than two months out with a knee problem, in the final warm-up clay-court tournament before the French Open.
Four-time Rome winner Serena, 37, had eased through 6-4, 6-2 her first round match against Swedish qualifier Petersen.
Serena had also played Petersen in the second round at the Miami Open in March before withdrawing with a knee injury.
Her withdrawal sends Venus Williams, the 1999 winner, through to a third round meeting with either Johanna Konta of Britain or American seventh seed Sloane Stephens.
Serena — now ranked 11th — has completed just eight matches this year, also withdrawing from Indian Wells in March with illness.
There are now doubts that the veteran can add to her tally of Grand Slams, something which has so far proved beyond her since returning last year after taking time out following the birth of her first child.
Her last Grand Slam singles title was won at the Australian Open in 2017, where she was pregnant, breaking a tie with Steffi Graf for the most in the professional era.
Her win in Melbourne moved her one behind Australian Court’s record of 24 major singles titles.
She is a three-time French Open winner, claiming her last title in Roland Garros in 2015.